Study On The Effect And Mechanism Of Artesunate On Proliferation,invasion And Metastasis Of Salivary Adenoid Cystic Carcinoma Cells Based On PI3K/Akt Pathway

Objective:To study the effect of artesunate（ART）on the proliferation,apoptosis,migration and invasion of salivary adenoid cystic carcinoma cells in vitro,and to determine whether ART has anti-tumor effect on adenoid cystic carcinoma cells through PI3K/Akt/mTOR signaling pathway and its possible mechanism.Methods:SACC-83 adenoid cystic carcinoma cells were treated with different concentrations of ART,and the related inhibitory effects of ART on SACC-83 cells were detected.1.Drug concentration screening test:SACC-83 adenoid cystic carcinoma cells were treated with ART at different concentration gradients of 25μmol/L,50μmol/L,100μmol/L,200μmol/L,400μmol/L,800μmol/L,1600μmol/L and 3200μmol/L,respectively.The SACC-83 cells were treated for 24 h,48 h and 72 h respectively.Then,CCK-8 cell proliferation/toxicity test was used to detect the cytotoxicity of the drug to the cells,and the following working concentration was selected according to the experimental results.2.The biological functions of ART on human adenoid cystic cancer cells were examined at different working concentrations.CCK-8 method was used to detect the cell proliferation.The ability of cell clone formation was detected by plate clone formation assay.The ability of cell migration was detected by cell scratch test.Transwell matrigel invasion assay was used to detect the change of cell invasion ability.Transwell migration assay was used to detect the change of cell longitudinal migration ability.3.Immunocytochemical staining was used to detect the change of protein expression level in SACC-83 of human adenoid cystic carcinoma cells treated with different drug concentrations.4.The expression levels of PI3K,Akt,mTOR and other related proteins in SACC-83 cells were detected by immunofluorescence staining.5.The expression levels of PI3K,Akt,mTOR and other related target genes in SACC-83 cells treated with different concentrations of ART for 48 h were detected by real-time fluorescence quantitative PCR（RT-QPCR）.6.Western Blot（WB）was used to detect the expression levels of PI3K,Akt and mTOR in SACC-83 cells.Results:1.CCK-8 test results showed that SACC-83 cell growth was significantly inhibited to different degrees under different ART concentrations,and the inhibition degree was concentration-dependent.The longer the intervention time under the same concentration,the stronger the inhibition effect was.The half maximal inhibitory concentration(IC₅₀)against SACC-83cells was（427.57±37.34）mol/L and the IC₅₀ was（61.36±17.54）mol/L.According to the set reference range of 1/4 IC₅₀ and 1/2 IC₅₀,15 mol/L,30mol/L,60 mol/L,120 mol/L and 240 mol/L were used as the working concentration of the experimental drug in subsequent experiments of ART,and the better intervention effect could be obtained when the treatment time was chosen at 48 h.2.The results of plate colony formation assay showed that the colony forming number of SACC-83 cells decreased significantly after ART treatment,and decreased with the increase of ART concentration,and the difference was statistically significant（P<0.05）.Compared with the control group,the number of cells in the SACC group was significantly lower than that in the control group（P<0.05）.The results of Transwell migration test showed that the number of SACC-83 cells in 15μmol/L ART group was significantly lower than that in blank control group,and with the increase of concentration,the number of SACC-83 cells migration decreased gradually in the same time,the difference was statistically significant（P<0.05）.Transwell matrix gel invasion test showed that the invasion number of SACC-83 cells after 15μmol/L ART intervention was significantly lower than that of normal cancer cells,and with the increase of the concentration,the invasion number of SACC-83 cells gradually decreased at the same time,the difference was statistically significant（P<0.05）.3.The results of immunocytochemistry showed that when the concentration of ART was 0μmol/L,the average optical density of cells was the highest when the concentration of ART was 0μmol/L;the protein expression of PI3K,Akt and mTOR decreased with the increase of drug concentration in other intervention groups,the difference was statistically significant（P<0.05）.4.qPCR was used to detect the expression of PI3K,Akt,p-Akt and mTOR genes in SACC-83 cells.The results showed that the relative m RNA expression of PI3K,Akt,p-Akt and mTOR decreased significantly with the increase of drug concentration under the same treatment time,and the difference was statistically significant（P<0.05）.5.Western blot results showed that the relative expression levels of PI3K,Akt,p-Akt and mTOR in SACC-83 cells treated with ART at different concentrations for 48 h were significantly lower than those in the untreated group（P<0.05）.Conclusions:1.ART can inhibit the proliferation,cloning,invasion and migration of human salivary gland adenoid cystic carcinoma cell line SACC-83 in vitro.2.Artesunate can effectively inhibit the PI3K/Akt/mTOR pathway in SACC-83 cells and down-regulate the molecular expression of key factors in the pathway,including PI3K,Akt and mTOR,and its inhibitory ability is drug concentration dependent.